Ubiquitin-Specific Protease2B Negatively Regulates IFN-βProduction And Antiviral Activity By Targeting TANK-Binding Kinase1

Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), Nod-like receptors (NLRs) and sensors of DNA, detect pathogen-associatedmolecular patterns (PAMPs) of microbes to initiate innate immune response. After sensing a PAMP, these PRRs trigger activation of the common transcription factors such as NF-κB, IRFs and AP-1, which leads to the production of proinflammatory cytokines, type I interferons and induction of subsequent adaptive immune responses. Type I IFNs interact with its receptors’ then activate the JAK-STAT signal transduction pathways, leading to induction many downstream IFN-stimulated genes (ISGs). The products of these ISGs collaborate to inhibit viral replication or eliminate of virus-infected cells, leading to subsequent antiviral responses. Therefore, type I IFNs play very important roles in host defense against invading viruses. Although type I interferon is required for viral clearance, aberrant production of type I interferon may promote the development of immunopathological conditions. Thus, tight regulation of type I interferon signaling is critical for maintaining the homeostasis of both innate and adaptive immunity. DUBs are proteases which specifically cleave the isopeptide bond at the C terminus of ubiquitin or ubiquitin-like proteins from target proteins. A few DUBs have been shown to regulate virus-induced type I interferon signaling. For example, DUBA、A20、USP3. USP4and USP25have been demonstrate to regulate type I IFN signaling through different molecular mechanims. USP2, a deubiquitinating enzyme, is a member of the USP family. So far at least three USP2isoforms have been identified due to alternative splicing, as follows: USP2-69(USP2a), USP2-45(USP2b), and USP2-41(USP2c). Recently, it has been demonstrated that USP2a negatively regulates IL-1β-and virus-induced NF-κB activation by deubiquitinating TRAF6, however, the functions of USP2, especially USP2b in the regulation of type I interferon signaling and antiviral immunity are largely unknown. Therefore, in this report we reveal the function and the molecular mechanism of USP2in regulating type I interferon signaling and antiviral immunity.Objectives:1. Determine the function of USP2in type I IFN signaling and antiviral immunity and find which isform will play a role in it.2. Find out the mechanism of USP2b in regulating in type I IFN signaling.3. Discuss the functions of USP2b in the other innate immune responses.Methods:1. Detect USP2expression in SeV-traggered type I IFN signaling HEK293or THP-1cells were infected with SeV, Western blot analysis of USP2protein expression.2. USP2b regulates virus-induced IFN-β expression2.1Detect the effect of USP2in SeV-induced IFN-β-luc activity by reporter assays. HEK293cells were transiently transfected with IFN-β-luc reporter plasmid together with increasing amount of USP2a, USP2b, and USP2c expression plasmids for20h, IFN-β luciferase activity was measured after treatment with SeV for8hHEK293cells were transfected with control siRNA or USP2siRNA, together with IFN-P-luc plasmid for48h, IFN-β luciferase activity was measured after treatmet with SeV for8h2.2RT-PCR analysis of IFN-β mRNA levelRT-PCR analysis of mRNA expression in HEK293cells transfected with USP2a, USP2b, and USP2c expression plasmids or control plasmid for20h, and then left untreated or treated with SeV for8h.HEK293cells were transfected with control siRNA or USP2siRNA, together with IFN-β-luc plasmid for48h, and then infected with or without SeV for8h, IFN-β luciferase activity was measured after untreated or treated with SeV for8h.3. Search the molecular mechanisms of USP2b in regulating in type I IFN signaling3.1Determine the target molecule of USP2bHEK293cells wrere transfected with RIG-I, MDA5, MAVS, TBK1, and IRF3-5D along with USP2b expression plasmid or control vector for24h, then RT-PCR analysis IFN-β mRNA expression.HEK293cells were transfected with RIG-I, MDA5, MAVS, TBK1, and IRF3-5D and IFN-β reporter plasmid along with USP2b expression plasmid or control vector, IFN-P-luc activity was measured.3.2Interact between USP2b and TBK1HEK293cells were transfected with Flag-USP2b along with HA-TBK1, cell lysates were IP with anti-Flag beads and immunoblot analysis with anti-HA Ab THP-1cells were infected with SeV for various time points, followed by IP with USP2Ab or control IgG and immunoblot analysis with anti-TBK1Ab.3.3Affect status of K63-linked TBK1ubiquitination by USP2bHEK293cells were transfected with Myc-TBK1, HA-Ub and Flag-USP2b plasmids or USP2siRNA, followed by IP with anti-Myc Ab and immunoblot analysis with anti-HA Ab.Myc-TBK1, Flag-USP2b, and HA-Ub (K48) or HA-Ub (K63) plasmids were transiently transfected in HEK293cells, followed by IP with anti-Myc Ab and immunoblot analysis with anti-HA Ab.HEK293cells transfected with Myc-TBK1HA-Ub and Flag-USP2b or USP2b C67A plasmids, followed by IP with anti-Myc Ab and immunoblot analysis with anti-HA Ab.3.4Affect the kinase activity of TBK1by USP2bHEK293cells were transfected with control siRNA or USP2siRNA, cells were infected with or without SeV for6h. Cell extracts were immunoprecipitated with anti-TBK1Ab, and TBK1kinase activity was measured4. Investigate the function of USP2b in cellular antiviral responseHEK293cells were transfected with the indicated plasmids for24h, cells were untreated or treated intracellular with poly (I:C) for8h. The cells were infected with VSV and the supernatants were harvested at12h after infection. Standard plaque assays analyze VSV titers of the supernatants. Quantitative RT-PCR analysis intracellular VSV RNA.HEK293or THP-1cells were treated with control siRNA or USP2b siRNA for36h. VSV titers and intracellular VSV RNA replicates were measured as above.5. Detemine the function of USP2b in the other innate immune responses USP2b plasmid or USP2siRNA, TRIF, cGAS plus STING, and IFN-β reporter plasmids were transfected into HEK293cells, IFN-P-luc activity was measuredTHP-1cells were transfected with USP2b siRNA or control siRNA for48h, then treated with LPS and poly (I:C), or transfected with ISD for12h, IFNβ mRNA expression was measured by RT-PCRResults:1. HEK293cells and THP-1cells were infected with SeV for different time points, USP2a and USP2b expression were reduced. However, USP2c was not detected in both cell types.2. USP2b negatively regulates virus-induced IFN-β expression.2.1Overexpression of USP2b inhibited SeV-induced IFN-β-luc activity.2.2Overexpression of USP2b inhibited SeV-induced IFN-β mRNA expression.2.3Overexpression of USP2b inhibited SeV-induced IRF3-luc activity.2.4USP2b C67A mutant lost the ability to inhibit SeV-induced IFN-β-luc activity and expression of IFN-β mRNA.3. Knockdown of USP2b enhances IFN-β expression.3.1USP2-specific siRNAs could efficiently knock down USP2b expression (mRNA and protein level).3.2Knockdown of USP2b could significantly enhance SeV-induced IFN-β-luc activation and expression of IFN-β mRNA.4. USP2b targets TBK1.RIG-I-, MDA5-, MAVS-, and TBK1-induced IFN-β-luc activity and IFN-β mRNA were strongly decreased when USP2b was overexpression, whereas IRF35D-induced IFN-β-luc activity and IFN-P mRNA were not impaired by USP2b overexpression.5. USP2b deubiquitinates K63-linked polyubiquitin of TBK1 5.1USP2b interacts with TBK15.2Ubiquitin of TBK1were greatly decreased by USP2b overexpression, however, USP2C67A lost the ability. In contrast, ubiquitination of TBK1were greatly enhanced when USP2b was knockdown.5.3Overexpression of USP2b significantly decreased K63-linked ubiquitination of TBKl.6.USP2b attenuates TBK1kinase activityOverexpression of USP2b could inhibit TBK1kinase activity in SeV infected HEK293cells, in contrast, knockdown of USP2b had opposite effect7.USP2b negatively regulates cellular antiviral responseOverexpression of USP2b increased VSV viral replication and VSV RNA replicates, in contrast, knockdown of USP2b had opposite effect.8. USP2b negatively regulates TLR3/4and DNA-induced IFN-β signalingOverexpression of USP2b substantially attenuated TRIF and cGAS plus STING-induced IFN-β promoter activation, in contrast, knockdown USP2increased IFN-β promoter activation. In THP1cells, IFN-β mRNA expression was increased after stimulation with LPS and poly (I:C), or transfected with ISD when USP2b was knockdown. Conclusion:1. Expression of USP2b was reduced in SeV infected cells, USP2b negatively regulates virus-induced type I IFN pathway.2. USP2b interacts with TBK1and deubiquitinates K63-linked polyubiquitination of TBK1.3. USP2b negatively regulates cellular antiviral responses.4. USP2b negatively regulates TLR3/4and DNA-induced IFN-P signalingInnovation and significances:1. In this report, we first demonstrated USP2b negatively regulates IFN-P production and antiviral activity by targeting TBK1. Further, we showed that USP2b specifically binded to TBK1and removed K63-linked polyubiquitination from TBK1, therefore, inhibited TBK1kinase activity.2. Given TBK1is essential for IRF3activation and IFN-β production down stream of various innate receptors, we demonstrated USP2b may represent a general regulator in virus-triggered IFN-β production and cellular antiviral responses.3. Our studies provided new experimental evidence for negative regulation of IFN-β production after viral infection. Therefore, USP2b may be used as a useful target for manipulating excessive innate immune response after viral infection.